Super-resolution Investigation of Synaptic Function
Final Report Abstract
The synapse has been studied for more than 60 years, using techniques spanning from electron microscopy to electrophysiology and advanced forms of live imaging. These works have provided a comprehensive view of cellular and organelle morphology. In contrast, protein localization and distribution within the cell has been much harder to investigate, especially in small specialized structures such as the synapse. Current tools are not optimal for such approaches: conventional light microscopy is limited by diffraction to spots of ~200- 300 nm; electron microscopy, while offering unsurpassed resolution, is not trivial to use in protein localization studies, as the different elements need to be identified by affinity labelling – which is much less efficient in electron microscopy when compared to light microscopy. We used here a diffraction-unlimited fluorescence imaging technique, Stimulated Emission Depletion (STED), to characterize the positions of the major components of the synapse. We determined the relative locations of synaptic proteins involved in neurotransmitter release, in membrane retrieval, in active zone structure, in endosomal recycling, and also of less specialized elements such as the cytoskeleton and cytosolic proteins. In addition, we answered questions on the fate of synaptic vesicles upon fusion to the plasma membrane, and on the involvement of endosomes in synaptic vesicle recycling In a parallel effort, we determined the copy numbers for a substantial number of these proteins (roughly 60). We are now using the knowledge given by the numbers and relative positions to generate a model of the synapse. The usefulness of this model is manifold. It gives for the first time all volumes (including volumes occupied by proteins) in the synapse and the locations and numbers of more than 60 proteins. It also compares two widely different synapses, from the central and peripheral systems. Therefore, it will be useful in allowing scientists to test in a realistic fashion the following: 1) Concepts related to the buffering of calcium. Knowing the exact volumes, as well as the numbers of proteic calcium buffers, will allow the modelling of calcium entry and buffering upon synaptic activity. 2) Concepts related to synaptic vesicle recycling. Clearly, knowledge on where the endocytosis machinery is located (and what numbers of molecules it contains) is vital in this enterprise. 3) Concepts related to the mobility and organization of vesicle pools. 4) General concepts related to the organization of cellular elements. Finally, the model of the synapse is the first realistic representation of a “piece” of a cell, in three dimensions, thus being of interest also in itself.
Publications
- (2010) Endosomal sorting of readily releasable synaptic vesicles. Proc Natl Acad Sci U S A 107:19055-19060
Hoopmann P, Punge A, Barysch SV, Westphal V, Bückers J, Opazo F, Bethani I, Lauterbach MA, Hell SW, Rizzoli SO
- (2010) Limited intermixing of synaptic vesicle components upon vesicle recycling. Traffic 11:800-812
Opazo F, Punge A, Bückers J, Hoopmann P, Kastrup L, Hell SW, Rizzoli SO
- (2011). Imaging synaptic vesicle recycling using FM dyes. In: Imaging in Neuroscience: A Laboratory Manual. Editors: Fritjof Helmchen, Arthur Konnerth. Cold Spring Harbor Laboratory Press
Hoopmann P, Rizzoli SO, Betz WJ
- (2012) FM dye photoconversion for visualizing synaptic vesicles by electron microscopy. Cold Spring Harb Protoc 2012:84-86
Hoopmann P, Rizzoli SO, Betz WJ
- (2012) Imaging synaptic vesicle recycling by staining and destaining vesicles with FM dyes. Cold Spring Harb Protoc 2012:77-83
Hoopmann P, Rizzoli SO, Betz WJ
